Immunofluorescence and immunohistochemistry for apoptosis.

Slides were deparaffinized with xylene and rehydrated with ethanol. Antigen retrieval was performed using Tris-EDTA buffer (pH 9). TUNEL staining was performed using the TUNEL Assay Apoptosis Detection Kit, CF 488A (Biotium), per manufacturer’s protocol. Nonspecific antigens were blocked with DAKO blocking solution (Agilent). Slides were incubated with anti–proSP-C antibody (1:2000, MilliporeSigma, AB3786) overnight at 4°C, followed by incubation with Alexa Fluor 648–conjugated chicken anti-rabbit secondary antibody (1:100, Life Technologies, Thermo Fisher Scientific, A21443) at room temperature for 1 hour and application of Vectashield mounting media with DAPI (Vector Laboratories). Images were captured with a Nikon Eclipse Ti microscope. Automated counting of total cells and proSP-C–positive cells was performed using ImageJ software functions “threshold” and “analyze particles,” with a size threshold of 2 μm² and circularity > 0.1. Immunohistochemistry for cleaved caspase-3 was performed using rabbit anti–cleaved caspase-3 (Asp175) (1:50, R&D Systems, Bio-Techne, MAB836). The slides were deparaffinized and rehydrated with distilled water. They were then placed in EDTA epitope retrieval buffer at 95°C for 30 minutes, then cooled and rinsed, and then placed in TBS with Tween. This is the same solution that was used in subsequent washing steps. Endogenous peroxidase was blocked using 3% hydrogen peroxide, then rinsed. The slides were then rinsed, and antibodies were detected with HRP-conjugated anti-rabbit secondary antibody. DAB was used to identify the reaction. Then the slides were washed and then counterstained in hematoxylin, dehydrated, cleared, and mounted with resinous mounting media. Images were acquired with a Nikon DS-Ri2 microscope.